1. M. Birkou, S. Bellou and G. Aggelis
Lipid composition of Penicillium sp. and Aspergillus niger cultivated on glycerol
M. Birkou, S. Bellou and G. Aggelis
Unit of Microbiology, Division of Genetics; Cell & Development Biology, Dpt of Biology; University of Patras; Greece
Introduction
Glycerol is a renewable resource appearing in increasing quantities as the principal by-product of fat saponification and alcoholic beverage fabrication units. One way of glycerol valorization is referred to its biotransformation to single cell oil (SCO) by oleaginous yeasts and moulds. Microbial lipids could contribute to the covering of the increasing demand of fats and oils, as in general, their structure and composition are similar to those of common vegetable oils [1]. Penicillium sp (two strains) and Aspergillus niger were cultivated on nitrogen-limited media having glycerol as sole carbon substrate, and changes in lipid composition were monitored during growth. Target of the present study was to investigate fatty acid distribution in the major lipids when glycerol was used as sole substrate in nitrogen-limited cultures [1].
Materials and Methods
Microorganisms: Penicillium sp., Aspergillus niger . Culture conditions: in 250 cc Erlenmeyer flasks containing 50 ml of a liquid medium Microorganisms were cultivated under nitrogen limited conditions and were incubated in a rotary shaker at T=28°C and 180 rpm. Medium: Glycerol at 25 g/l, supplemented with minerals and yeast extract. HPLC analysis: Glycerol was determined in filtered (through 0.2 μm pore size bacteriological filter, Whatman) aliquots of the culture by an HPLC apparatus (Ultimate 3000, Dionex, Germering, Germany) equipped with an HPX-87H column and a R.I. detector. Conditions: eluant H2SO N, flow rate 0.9 ml/min, T =55 oC. Lipid extraction: According to Folch protocol [2]. Lipid fractionation: by using a column of silicic acid activated by heating overnight at 80 °C. Successive applications of dichloromethane, acetone and methanol produced fractions containing neutral lipids (NL), glycolipids plus sphingolipids (G+S) and phospholipids (P), respectively [3]. GC analysis: Fatty acid analysis was performed after trans-methylation according to the AFNOR method [4], in an Agilent Technologies 7890 A device equipped with a HP-88 (J&W scientific) column (60 m x 0.25 mm). Conditions: carrier gas helium, flow rate 1 ml/min, oven T=200 °C, injector T=250 °C, detector (FID) T=280 °C.
Results and Discussion
Changes in lipid composition of Penicillium sp. (two strains, A3 and A6) and Aspergillus niger were monitored during growth under N-limited conditions. Lipid analysis showed that at the beginning of growth the mycelium was rich in neutral lipids (NL), in Penicillium sp.(A3, 79,70 % wt/wt oil in dry biomass and A6, 60 % wt/wt oil in dry biomass ) (Fig.1,2) and in Aspergillus niger (82 % wt/wt oil in dry biomass) (Fig.3), which indicated that most of the accumulated lipid proceeds as reserve lipid, while polar lipids [phospholipids, (P), glycolipids and sphingolipids (G+S)] were synthesized in limited amounts. Fatty acid composition showed significant changes with time. Linoleic acid (C 18:2) was the principal fatty acid in all lipid fractions and showed significant changes during fungal growth as well. Specifically, a decrease linoleic acid concentration was noticed in P fraction (Table 1,2 ). Linoleic acid synthesized in P fraction and then it migrated to the other lipids fractions (NL and G + S). Exceptionally, in A. niger linoleic acid was probably synthesized at early stages of growth and migrated in other lipids. For instant P fraction contained linoleic acid at percentage comparable to that observed in NL fraction, after 48 h cultivation (Table 3).
Fungi
t (h)
lipid fraction
C16:0
C16:1
C18:0
C18:1
C18:2
C18:3
others
Penicillium sp. A3 48 NL
13,75
2,41
6,46
19,35
52,44
3,84
1,56
G+S
15,35
4,36
7,89
17,47
49,55
2,86
2,72 P
13,28
5,15
5,00
15,14
57,43
2,63
1,37 96
13,17
1,12
4,55
22,46
55,42
2,15
1,13
18,38
1,72
5,94
19,26
47,44
3,57
3,70
13,48
2,01
2,59
19,34
57,90
1,75
2,93
168
14,67
0,93
7,47
24,13
49,61
1,73
1,46
19,40
2,90
3,97
21,38
46,00
2,04
3,72
14,56
0,58
2,99
20,66
55,91
2,14
3,15
Table 1: Fatty acid composition of NL, G+S and P lipid fractions during growth of Penicillium sp. A3
Fungi
t (h)
lipid fraction
C16:0
C16:1
C18:0
C18:1
C18:2
C18:3
others
Penicillium sp. A6 48 NL
14,09
1,03
6,26
19,37
55,25
2,00
G+S
20,34
6,00
11,16
15,54
42,59
1,75
2,61 P
15,42
1,09
5,85
12,42
60,94
2,38
1,89 96
12,87
0,79
5,16
19,72
58,22
1,59
1,64
18,42
1,00
6,51
18,51
49,97
2,08
3,51
14,67
6,24
4,12
57,63
1,08
3,38
168
16,53
1,06
8,91
24,97
43,67
1,96
2,91
22,25
1,68
10,28
23,72
36,52
1,85
3,7
1,05
4,91
20,20
54,59
2,21
1,61
Figure 1: Kinetic of growth Penicillium sp. A3
 Biomass X (g/l), Glycerol Glol (g/l), Lipids L (g/l)
Table 2: Fatty acid composition of NL, G+S and P lipid fractions during growth of Penicillium sp. A6
Fungi
t (h)
lipid fraction
C16:0
C16:1
C18:0
C18:1
C18:2
C18:3
others
Aspergillus niger 48 NL
17,88
1,05
9,33
21,60
43,35
5,59
1,21
G+S
24,67
3,73
16,18
20,94
29,83
3,65
1,00 P
19,56
6,86
10,30
21,37
41,08
5,83
1,01 96
17,97
1,04
13,46
29,70
33,54
3,09
1,36
17,47
0,39
12,26
30,51
32,93
4,81
1,63
17,44
0,78
10,61
29,81
36,75
1,52
168
12,34
0,70
6,44
24,65
50,88
3,18
1,80
15,37
0,79
9,36
28,55
40,79
2,44
2,69
10,73
2,03
11,18
24,95
45,36
2,5
3,26
Figure 2: Kinetic of growth Penicillium sp. A6
 Biomass X (g/l), Glycerol Glol (g/l), Lipids L (g/l)
Table 3: Fatty acid composition of NL, G+S and P lipid fractions during growth of A. niger
References
1. S., Papanikolaou, G., Aggelis, (2002). Lipid production by Yarrowia lipolytica growing on industrial glycerol in a single-stage continuous culture, Bioresource Technology 82,
2. Folch et al. (1957). A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 199:833–841
3. Fakas et al. (2006). Lipids of Cunninghamella echinulata with emphasis to g-linolenic acid distribution among lipid classes. Appl Microbiol Biotechnol., 73, 676–683.
4.AFNOR (1984) Recueil des normes francaises des corps gras, grains oléagineux et produits dérives. In: Association Française pour Normalisation. Paris, p 95
Acknowledgments
Financial support was provided by the project “ Biodiesel production from agricultural by- products” funded by the Greek fuel company, Dracoil SA.
Figure 3: Kinetic of growth A. niger
 Biomass X (g/l), Glycerol Glol (g/l), Lipids L (g/l)